Hematopoietic stem cells (HSCs) persist throughout life and dynamically regulate their numbers after injury by undergoing self-renewing divisions that depend upon both cell- intrinsic and cell-extrinsic mechanisms. With respect to cell-extrinsic mechanisms, HSCs are thought to reside within specialized microenvironments created by supporting cells in the bone marrow that express membrane-bound and secreted factors that promote HSC maintenance (survival and self-renewal), and that regulate HSC migration, quiescence, and differentiation. Many bone marrow HSCs reside at, or near, the osteoblasts at the endosteal surface and osteoblasts have been proposed to secrete a number of factors that promote HSC maintenance. Many HSCs also reside adjacent to sinusoidal blood vessels in the bone marrow, and vascular or perivascular cells have also been proposed to secrete factors that regulate HSC maintenance. Nonetheless, none of these factors have ever been conditionally deleted from any candidate niche cell. As a result, the physiological sources of these factors have never been tested in functional experiments. Angiopoietin-1 (Ang-1), Stem Cell Factor (SCF), and CXCL12 are all genetically required for maintenance of normal numbers of HSCs but none of these factors have been conditionally deleted from osteoblasts or from vascular/perivascular cells to identify the biologically important source(s) of these factors. Ultimately, it will not be possible to identify the cells that create HSC niches without genetically identifying the cells that secrete factors required for HSC maintenance. Our preliminary expression data suggest that megakaryocytes are the major source of Ang-1 in the bone marrow and that there are multiple sources of SCF including both endothelial cells and endosteal cells. To test which cells are functionally important sources of these factors for HSC maintenance we have generated floxed alleles of Ang-1 and Scf and propose to mate mice bearing these alleles with mice expressing Cre-recombinase under the control of promoters specific to osteoblasts, megakaryocytes, and endothelial cells. These experiments will also test whether a single cell type is the main source of multiple factors required for HSC maintenance or whether different cell types produce different factors that regulate HSCs. This will provide the first functional test of which cells regulate HSC maintenance in vivo. PUBLIC HEALTH RELEVANCE: This project is designed to assess which cells are the physiologically important sources of growth factors that are critical for the maintenance and regulation of hematopoietic stem cells. This will provide important new information regarding the identities of the cells that constitute the hematopoietic stem cell niche in vivo. Such information is critical to understand how blood cell formation is regulated and how stem cells are sustained in this tissue throughout life.